CN1378560A - Compounds and methods of use for treating infectious diseases and disorders of the immune system - Google Patents

Abstract

The present invention provides polypeptides comprising immunogenic epitopes of Mycobacterium vaccae proteins, polynucleotides encoding the polypeptides, fusion proteins comprising at least one of the polypeptides, and DNA constructs comprising at least one of the polynucleotides of the present invention. Compositions comprising the polypeptides, polynucleotides, fusion proteins, and/or DNA constructs can be used to treat infectious and immune diseases.

Description

The compound and the using method thereof of treatment infectious diseases and disease of immune system

Technical field

The present invention relates generally to detection, treatment and prevention to infectious diseases.Particularly, the present invention relates to contain the compound of separation from the immunogenicity epi-position of cow mycobacterium (Mycobacterium vaccae), and relate to these compounds aspect the vaccine inoculation of anti-infectious disease or immunotherapy and in the purposes aspect some Immunological diseases and the cancer therapy, described infectious diseases comprises mycobacterial infections, infects or mycobacterium avium (Mycobacterium avium) infection such as mycobacterium tuberculosis (Mycobacteriumtuberculosis).

Background of invention

The present invention relates generally to treatment and prophylaxis against infection diseases, and treat some Immunological diseases and cancer.Particularly, the present invention relates to treatment and the prevention mycobacterial infections Compounds and methods for of (comprising that mycobacterium tuberculosis or mycobacterium avium infect).

Tuberculosis is a kind of chronic infectious disease that causes that infected by mycobacterium tuberculosis (M.tuberculosis).Tuberculosis is a kind of principal disease in developing country, and also more serious in developed regions, world problem, annual about 8,000,000 newly-increased cases, 3,000,000 people's death.Although m tuberculosis infection may be asymptomatic in the quite a long time, the most normal chronic pneumonia that shows as of this disease causes heating and Respiratory symptoms.If do not treat, can cause the state of an illness significantly to increase the weight of even death.

Although generally prolong antibiotic therapy may command tuberculosis, such treatment is not enough to prevent spreading of this disease.Infected individual possibility is asymptomatic, but within a certain period of time can contagious infection.In addition, although very crucial to the compliance of treatment plan, patient's behavior is difficult to monitoring.Some patient does not finish the course of treatment, and this may cause failing to respond to any medical treatment and developing immunity to drugs mycobacterium.

Suppress that the tuberculosis diffusion needs effective vaccine inoculation and to the correct early diagnosis of disease.At present, the live bacterial vaccines inoculation is the most effective method of inducing protective immunity.The mycobacterium of normal use is bacille Calmette-Guerin vaccine (BCG) for this reason, a kind of Mycobacterium bovis (Mycobacteriumbovis) avirulent strain.Yet the security of BCG and validity have arguement always, and national as the U.S. at some, do not inoculate general crowd.Usually use skin test diagnosis m tuberculosis infection, skin test is exactly intracutaneous contact tuberculin PPD (purified protein derivative).T cells with antigenic specificity is replied after injection and to be produced detectable scleroma, indication contact antigen of mycobacterium thus in 48-72 hour in the injection site.Yet skin test has susceptibility and specific problem, and can not distinguish BCG inoculation individuality and infected individuals.

The cow mycobacterium is a kind of few well known mycobacterium that has been used for immunotherapy tuberculosis and leprosy, is non-pathogenic agent to the people.Yet, compare with BCG, less about the information of cow mycobacterium validity, and it also is not widely used in the general crowd of inoculation.Mycobacterium bovis BCG and cow mycobacterium are considered to contain the antigenicity compound of the individual immunity system identification that contacts m tuberculosis infection.

Some patents and other publication disclose by giving mycobacterium (comprising the cow mycobacterium) or the various illnesss of some mycobacterium component for treating.International Patent Publication No. WO 91/02542 discloses the treatment of chronic inflammation disease that patient is wherein discharged unusually high-level IL-6 and/or TNF or patient's a high proportion of non-semi-lactosi IgG of IgG display abnormality.Psoriasis, rheumatoid arthritis, mycobacterium disease, Crohn disease, primary biliary cirrhosis, sarcoidosis, ulcerative colitis, systemic lupus erythematous, multiple sclerosis disease, Guillain-Barre syndromes, primary diabetes mellitus and the graft-rejection aspect some are arranged in the middle of the disease of mentioning in the disclosure patent.Medicine preferably includes the single dose injection and gives autoclaved cow mycobacterium.

United States Patent (USP) 4,716,038 discloses to the diagnosis of (comprising sacroiliitis) of various types of autoimmune diseases, specific aim vaccine inoculation with by giving mycobacterium and has comprised that the cow mycobacterium treats.United States Patent (USP) 4,724,144 disclose and have been used for the treatment of mycobacterium disease especially tuberculosis and leprosy and as the immunotherapeutic agent of chemotherapy auxiliary, and it comprises the antigenicity substance that derives from the cow mycobacterium.International Patent Publication No. WO 91/01751 discloses uses the antigenicity substance that derives from the cow mycobacterium and/or immune regulator as the immunoprophylaxis agent, to postpone and/or prevention AIDS takes place.International Patent Publication No. WO 94/06466 discloses uses the antigenicity substance and/or the immune regulator treatment that derive from the cow mycobacterium to suffer from or do not suffer from AIDS and dependency HIV infection lungy.

Traditional vaccine comprises the pathogenic organisms (or its component) of attenuation or deactivation form.As the alternative method of traditional vaccine, developed dna vaccination at multiple disease such as AIDS, influenza, cancer and malaria.Clinical experiment well afoot at the dna vaccination of numerous such diseases.Typical dna vaccination comprises the DNA that is cloned into the coding for antigens in the non-activity plasmid vector.Usually be under the strong promoter control by the antigenic expression of described vaccine dna encoding, as people's beta-actin promotor, Rous sarcoma virus (RSV) promotor or CMV promotor (Ramsay AJ etc., Immunology and Cell Biology 75:360-363,1997).People such as Tang have obtained first experimental evidence (Tang D-C etc., Nature 356:152-154,1992) of the immunne response that dna vaccination can derived need.In these experiments, produce the specificity primary antibody response with the plasmid inoculation mouse that comprises the human growth hormone encoding gene.

In animal model, estimated the immunne response that contains the dna vaccination of mycobacterium tuberculosis gene at two kinds.First kind of vaccine contains GroEL stress protein (65 kDa albumen; Tascon RE etc., Nature Med.2:888-892,1996) encoding gene.The protection level that obtains with this dna vaccination injection mouse equates with the mouse of accepting traditional B CG vaccine.Second kind of dna vaccination at mycobacterium tuberculosis comprises the antigenic DNA of coding for antigens 85 mixtures, obtained the analog result (Huygen K etc., NatureMed.2:893-898,1996) with people such as Tang research.United States Patent (USP) 5,736,524 disclose by giving a kind of polynucleotide Vaccinum Calmette-Guerini immunization domesticated mammal or domestic animal, infect with opposing mycobacterium tuberculosis or Mycobacterium bovis, and described vaccine comprises antigen of mycobacterium tuberculosis 85 genes that effectively are connected to transcription regulatory element.

Reported first people's dna vaccination experiment (Wang R etc., Science 282:476-80,1998) recently.In this experiment, virulence factor plasmodium falciparum (Plasmodium falciparum) antigen of malaria is injected into healthy premenopausal volunteers.According to cytotoxic T (CD8 occurring ⁺) lymphocyte (CTL) confirmed to have excited the immunne response that needs, the prompting immunity system should be removed the intravital parasite of infected patient.The measuring that McGregor etc. carry out the security and the immunogenicity (J.Infect.Dis.178:92-100,1998) of people's dna vaccination of infecting of anti-HIV-1.The experimental data of other dna vaccination experiment is also pointed out, and produces the restricted CD8 of MHC I class behind the injection dna vaccination ⁺CTL and the restricted CD4 of MHC II class ⁺Helper cell antibody (Ramsay, AJ etc., Immunology and Cell Biology75:360-363,1997).

Dna vaccination significantly is better than comprising the multiple traditional vaccine of deactivation or attenuated organism.Dna vaccination is induced long-acting immunne response, and therefore may only need once inoculation.The multiple antigenic DNA of coding can be joined in the single plasmid, the protection of anti-multiple disease is provided thus.The technology of producing dna vaccination is simple relatively, and identical technology can be used for producing all vaccines, makes production cost more cheap.Effective traditional vaccine is passed to the patient depends on complete " the cold conveyer chain " of maintenance from the manufacturer to the clinic.Solution or the drying type dna vaccination produced are insensitive to condition of storage.

Developed the alternative route that makes up and provide dna vaccination recently.In a kind of therein technology that is called somatocyte transgenosis immunity (STI), the plasmid DNA direct inoculation that carries immunoglobulin heavy chain gene, be under the control of tissue specificity regulatory element is gone into mouse spleen, subsequently at the described antigen of B cell surface expression (Xiong S etc., Proc.Natl.Acad.Sci.USA94:6352-6357,1997).These B cells produce at expressed antigenic antibody, cause immunne response.Studies show that afterwards, the memory of STI inductive lasting immunity can reach 2 years (Gerloni M etc., Vaccine (2-3): 293-297,1998).

Expression library immunization (ELI) is the another kind of technology (Barry MA etc., Nature 377:632-635,1995) of using dna vaccination.In this technology, the fragment cloning of complete pathogen gene group is gone in the carrier also as vaccine.Select clone bank by screening and rescreening,, select protective antigen, especially induce the protective antigen of CTL until identifying single clone.Identified polynucleotides or polypeptide can be incorporated in the transfer system of approved use then.

(San Diego, scientist CA) is used for the treatment of and prevents progress (Ishioka GY etc., Journalof Immunology 162:3915-3925,1999) aspect the epitype vaccine that HIV infects in exploitation to disclose Epimmune Inc. recently.

The Compounds and methods for that this area still needs effectively prevention and treats infectious diseases (for example tuberculosis of people and domesticated mammal or domestic animal and other mycobacterial infections) and effectively treat some immunity system relative disease.

The invention summary

In brief, the invention provides the Compounds and methods for of prevention and treatment infectious diseases (as mycobacterial infections) and treatment Immunological diseases and cancer.

First aspect the invention provides and derives from the genomic separation polynucleotide of cow mycobacterium.The polypeptide epitope that the immunogenicity characteristic that these polynucleotide encodings are illustrated according to its panimmunity measurement result is selected.In specific embodiment, polynucleotide of the present invention comprise and are selected from following sequence: (a) sequence that provides among the SEQ ID NO:8-21; (b) measure according to computerized algorithm BLASTN, have the sequence of at least 50%, 75% or 90% identical residue with the sequence of SEQ ID NO:8-21; (c) sequence (a) and complement (b).

Second aspect the invention provides the isolated polypeptide of the immunogenicity epi-position that contains the cow antigen of mycobacterium.In specific embodiment, polypeptide of the present invention comprises and is selected from following sequence: (a) sequence that provides among the SEQ ID NO:61-77; (b) measure, have the sequence of at least 50%, 75% or 90% identical residue with the sequence of SEQ ID NO:61-77 according to computerized algorithm FASTX.

The DNA that contains at least a polynucleotide of the present invention also is provided construction, and transforms or the host cell of transfection with such DNA construction.

On the other hand, the invention provides the fusion rotein that comprises at least a polypeptide of the present invention.

About others, the invention provides the composition that comprises acceptable carrier at least a polypeptide of the present invention, polynucleotide, fusion rotein or DNA construction and a kind of physiology.The present invention also provides the composition that comprises at least a aforementioned polypeptides, polynucleotide, fusion rotein or DNA construction and a kind of immunologic stimulant.

Again on the one hand, provide the method that strengthens patient's immunne response, comprise one or more above-mentioned compositions that give patient's significant quantity.In one embodiment, described immunne response is that Th1 replys.

Another aspect of the present invention provides the method for the treatment of patient disease, comprises giving the patient composition of the present invention.In certain embodiments, described disease is selected from Immunological diseases, infectious diseases and cancer.

These aspects of the present invention and others can become more clear with reference to following detailed description the time.All be attached to separately herein as each reference, all reference disclosed herein integral body by reference are attached to herein.

The accompanying drawing summary

Figure 1A-F graphic extension reduces detection inducing protective immunity with Mycobacterium bovis BCG (being respectively Figure 1A and D), ME/DDNA (being respectively Figure 1B and E) or rME/D (being respectively Fig. 1 C and F) immunization BALB/cByJ mouse with mycobacterium tuberculosis CFU in its lung and the homogenation of spleen tissue.

The proliferative response of its lymph-node cell when Fig. 2 A-D shows with the subcutaneous immune BALB/cByJ mouse of rME/A (Fig. 2 A), rME/B (Fig. 2 B) or rME/D (Fig. 2 C).Control mice PBS immunity (Fig. 2 D).

The IFN-γ secretion of its lymph-node cell when Fig. 3 A-D shows with the subcutaneous immune BALB/cByJ mouse of recombinant multi-epitope construction rME/A (Fig. 3 A), rME/B (Fig. 3 B) or rME/D (Fig. 3 C).Control mice PBS immunity (Fig. 3 D).

The proliferative response of its lymph-node cell when Fig. 4 A shows with rME/A, rME/D or ME/D DNA by three kinds of different immunization routes immunity BALB/cByJ mouse.Fig. 4 B shows the proliferative response with the lymph-node cell of the control mice of PBS immunity.

The level of its lymph-node cell secretion of gamma-IFN when Fig. 5 A shows with rME/A, rME/D or ME/D DNA by three kinds of different immunization routes immunity BALB/cByJ mouse.Fig. 5 B shows the level with the control mice secretion of gamma-IFN of PBS immunity.

When Fig. 6 A showed with rME/A, rME/D or ME/D DNA by three kinds of different immunization routes immunity BALB/cByJ mouse, each epi-position was to the influence of mouse lymph nodal cell proliferative response.Fig. 6 B shows the proliferative response with the lymph-node cell of the control mice of PBS immunity.

When Fig. 7 A showed with rME/A, rME/D or ME/D DNA by three kinds of different immunization routes immunity BALB/cByJ mouse, each epi-position was to the influence of mouse lymph nodal cell secretion of gamma-IFN level.Fig. 7 B shows the level with the control mice secretion of gamma-IFN of PBS immunity.

The titre and the hypotype of anti--ME antibody in its serum when Fig. 8 A and B show the ME/D dna immunization mouse of using with rME/A and rME/D vitro reactions.Fig. 8 A shows the titre of IgG1 antibody, and Fig. 8 B shows the titre of IgG2a antibody.

IFN-γ secretion of its memory splenocyte when Fig. 9 A-C has shown with the immune BALB/cByJ mouse of reorganization single epi-position (Fig. 9 B) or rME/D (Fig. 9 C).Fig. 9 A shows the IFN-γ secretion with splenocyte after the control stimulation.

Figure 10 A and B have shown IFN-γ secretion (Figure 10 A) and the proliferative response (Figure 10 B) with human PBMC after rME/A, rME/B or the rME/D stimulated in vitro.

Figure 11 A and B have shown IFN-γ secretion (Figure 11 A) and the proliferative response (Figure 11 B) with human PBMC after eight kinds of single epi-position stimulated in vitro of reorganization.

Detailed Description Of The Invention

As mentioned above, the present invention relates generally to composition and the method for prevention and treatment disease (comprising infectious diseases and some immunological diseases and cancer). The example of such disease includes but not limited to mycobacterial infections, comprises that m tuberculosis infection and mycobacterium avium infect; And stimulate the useful disease of Th1 immune response, include, but is not limited to psoriasis and allergic rhinitis.

Some pathogen (such as Much's bacillus) and some cancer can be referred to as the CD4 of cell-mediated immunity⁺The immune attack of T cell control is effectively contained. The antibody that other pathogen such as poliovirus also need the B cell to produce is contained. These of immune attack dissimilar (T cell or B cells) are by different CD4⁺The control of T cell subsets is referred to as Th1 cell and Th2 cell usually.

This Th cell subsets of two types in the muroid model by characterization mistake fully, and the cell factor definition that when activating, is discharged by their. The secretion of Th1 subgroup IL-2, IFN-γ and TNF, and mediation macrophage activation and delayed hypersensitivity. The Th2 subgroup discharges IL-4, IL-5, IL-6 and the IL-10 that stimulates the B cell activation. Th1 subgroup and Th2 subgroup suppress mutually, reply so that IL-4 suppresses the Th1 type, and IFN-γ inhibition Th2 type are replied. Found similar Th1 subgroup and Th2 subgroup the mankind, the cell factor that their discharge with observe at the muroid model identical. It is the decisive factor that the numerous disease patient's condition reverses that Th1 type immune response strengthens, and described disease comprises respiratory disease, such as tuberculosis, sarcoidosis, asthma, allergic rhinitis and lung cancer.

On the one hand, composition of the present invention comprises polypeptide or its variant of the immunogenicity epi-position that contains at least a mycobacterium vaccae antigen. In specific embodiment, polypeptide of the present invention comprises the sequence that SEQ ID NO:61-77 provides. Such polypeptide stimulates T cell proliferation and/or the secretion IFN-γ of the individuality that has contacted Much's bacillus.

Term used herein " polypeptide " comprises the amino acid chain of random length, comprises full-length proteins (being antigen), and wherein said amino acid residue connects by the covalency peptide bond. Therefore, the polypeptide that contains the immunogenicity epi-position of above a kind of antigen can be comprised of described immunogenicity epi-position fully, perhaps can comprise other sequence. Described other sequence can derive from natural mycobacterium vaccae antigen or can be the heterologous sequence, and described sequence can (but not needing) be the immunogenicity sequence.

" immunogenicity " used herein refers to excite the ability of patient (such as the people) or biological sample immune response. Particularly, the immunogenicity epi-position be can the stimulating organism sample cell propagation, produce IL-12 or produce the polypeptide portion of IFN-γ, wherein said biological sample contains one or more following cells: the T cell, NK cell, B cell and the macrophage that derive from the mycobacterium immune body. In general, the determination techniques of using following examples 1 to describe in detail detects, and the immunogenicity epi-position stimulates the level of mycobacterium immune body PBMC propagation higher at least 2 times than what observe in contrast PBMC. Perhaps, or go back simultaneously, the ELISA mensuration OD that describes in detail according to embodiment 1 increases at least 2 times testing result, and the level that the PBMC of immunogenicity epi-position stimulation mycobacterium immune body produces IFN-γ is higher at least 2 times than what observe in control cells. The mycobacterium immune body is considered to utilize the effective t cell response for Much's bacillus, environment saprophage or BCG of having set up to resist the individuality that mycobacterial infections occurs. Such individuality can react according to strong positive (namely greater than about 10mm diameter scleroma) the intracutaneous skin test to tuberculoprotein (PPD) and not have any symptom of tuberculosis infection to identify. The polypeptide that contains at least a immunogenicity epi-position of one or more mycobacterium vaccae antigens generally is used in induces antituberculotic protective immunity and/or in patient's moderate stimulation immune response among the patient.

On the other hand, composition of the present invention comprises the separation polynucleotides of the immunogenicity epi-position of coding mycobacterium vaccae antigen. In specific embodiment, polynucleotides of the present invention comprise the sequence of SEQ ID NO:5-21. Complement, reverse complemental thing and the reverse sequence and their variant that also provide the present invention to separate polynucleotides. The present invention also comprise different from disclosed sequence but since genetic code degeneration and with the polynucleotide sequence of polynucleotide sequence coding phase homopolypeptide disclosed herein.

Term used herein " polynucleotides " refers to strand or the deoxyribonucleotide base of two strands or the polymer of ribonucleotide base, comprise dna molecular and the corresponding RNA molecule (comprising HnRNA and mRNA molecule) of sense strand and antisense strand, and comprise cDNA, genomic DNA and recombinant DNA and all or part of synthetic polynucleotides. The HnRNA molecule comprises introne and corresponding with dna molecular in common one to one mode. The mRNA molecule is corresponding to the HnRNA molecule and/or removed the dna molecular of described introne. Polynucleotides can be comprised of complete genome, or are comprised of its arbitrary part. Effectively antisense polynucleotides can comprise the fragment of corresponding polynucleotides, and therefore the definition of " polynucleotides " comprises all these effective antisense fragments. Antisense polynucleotides and the technology that relates to antisense polynucleotides are well-known in the art, are described in such as Robinson-Benion etc., " antisense technology ", Methods in Enzymol.254:363-375,1995; With Kawasaki etc., Artific.Organs 20:836-848,1996.

Following example has the most clearly been set forth the definition of term used herein " complement ", " reverse complemental thing " and " reverse sequence ". For sequence 5 ' AGGACC 3 ', complement, reverse complemental thing and reverse sequence are as follows:

Complement 3 ' TCCTGG 5 '

Reverse complemental thing 3 ' GGTCCT 5 '

Reverse sequence 5 ' CCAGGA 3 '.

Use these terms the same with common this area, all polynucleotide described herein and polypeptide all are separation and purification.

The compositions and methods of the invention also comprise the varient of aforementioned polypeptides and polynucleotide.Varient can be naturally occurring allelic variation body, perhaps can the naturally occurring varient of right and wrong.Term used herein " varient " comprise have at least about 30% with sequence of the present invention, more preferably at least about 50%, more more preferably at least about 75% with most preferably at least about arbitrary sequence of 90% identical residue (or Nucleotide or amino acid).By two comparative sequences being carried out the sequence contrast, measure the identical residue number of contrast part, will count residue sum, and the result be multiply by 100 divided by the present invention's (or inquiry) sequence, can record the percentage of identical residue.

Can carry out the sequence contrast to polynucleotide or peptide sequence, and can use the computerized algorithm that can openly obtain to measure in the concrete zone identical Nucleotide percentage with respect to another kind of polynucleotide.Two kinds of contrasts and identify that the typical algorithm of polynucleotide sequence similarity is BLASTN algorithm and fasta algorithm.Can use BLASTP and FASTX algorithm to detect the similarity of peptide sequence.BLASTN and BLASTP software all can the NCBI anonymous file transfer protocol server (ftp: //on ncbi.nlm.nih.gov) /obtain under the blast/executables/.BLASTN algorithm 2.0.6 version [9-16-1998] is preferred for measuring varient of the present invention, and the set default parameters of this algorithm is described in file and provides and delivers with this algorithm.Using of BLAST algorithm family (comprising BLASTN and BLASTP) is described in NCBI Web website and the publication Altschul of URL as http://www.ncbi.nlm.nih.gov/BLAST/newblast.html., Stephen F. etc., " room BLAST and PSI-BLAST: protein database search program of new generation ", Nucleic Acids Res.25:3389-3402,1997.Computerized algorithm FASTA can be on Internet ftp website ftp: //ftp.virginia.edu/pub/fasta/ obtains.The FASTA and the FASTX optimal algorithm selection of the 3.1t11 version in August, 1998 are used to measure varient of the present invention, and the set default parameters of this algorithm is described in file and provides and delivers with this algorithm.The use of fasta algorithm is described in for example Pearson, WR and Lipman, DJ, Proc.Natl.Acad.Sci.USA 85:2444-2448,1998; And Pearson, WR., Methods in Enzymol 183:63-98,1990.The use of FASTX algorithm is described in for example Pearson, WR etc., Genomics 46:24-36,1997.

Following working parameter (E value and identity percentage are provided) is preferred for using BLASTN to measure contrast and similarity: Unix action command: blastall-p blastn-dembldb-e 10-G 0-E 0-r 1-v 30-b 30-i queryseq-0 results; And parameter default is :-p program name [String];-d database [String];-e desired value (E) [Real]; Open (open) point penalty (0 calls default parameters) [Integer] in-G room;-E room prolongs point penalty (0 calls default parameters) [Integer]; The award (only for BLASTN) [Integer] of-r Nucleotide coupling; The number (V) [Integer] that-v single file is described;-b shows contrast number (B) [Integer];-i inquiry file [File In];-o BLAST report output file [File Out] is optional.For BLASTP, the working parameter below preferred use the: blastall-p blastp-dswissprotdb-e 10-G 0-E 0-v 30-b 30-i queryseq-0 results;-p program name [String];-d database [String];-e desired value (E) [Real]; The open point penalty (0 calls default parameters) [Integer] in-G room;-E room prolongs point penalty (0 calls default parameters) [Integer]; The number (V) [Integer] that-v single file is described;-b contrast of display number (B) [Integer];-I inquiry file [File In];-o BLAST report output file [File Out] is optional.

For using FASTX to measure contrast and similarity, preferred following UNIX order: fastx-E 10-b 30-H queryseq＞output, and for FASTA, preferred following UNIX order: fasta-E 2-b 30-H-n queryseq＞output.

" hit rate " of one or more database sequences contrasted and identifies the similar part of sequence by the search sequence of BLASTN, BLASTP, FASTA, FASTX or the acquisition of similar algorithm.According to similarity and sequence overlap length series arrangement hit rate.To the hit rate of database sequence representative overlapping on a part of sequence length of search sequence only usually.

BLASTN and fasta algorithm also produce sequence correlated " expection " value.When desired value (E) is illustrated in the database of retrieving a certain size, for the continuous sequence of certain number, the number that hits sequence that people's " expection " can find at random.Desired value is used as mensuration and whether the hit rate of database (such as preferred EMBL database) is represented the important threshold value of true similarity.For example, distributing to the E value of hitting sequence is 0.1, and its implication may be interpreted as in the database of an EMBL database size, and for the contrast part of the sequence with similar marking (score), people's expection only can obtain 0.1 matching sequence at random.According to this standard, the contrast of the described sequence probability identical with compatible portion is 90%.For being sequence below 0.01 or 0.01 in contrast and compatible portion E value, use BLASTN or fasta algorithm find at random that in the EMBL database probability of a matching sequence is 1% or littler.

According to an embodiment, " variation " polynucleotide with respect to each polynucleotide of the present invention preferably contain such sequence: it is compared with polynucleotide of the present invention, the nucleic acid number that contains is all identical with each polynucleotide of the present invention or slightly many, and the E value that produces is below 0.01 or 0.01.Be that the varient polynucleotide are following arbitrarily sequences: its probability identical with polynucleotide of the present invention is at least 99%, and using the BLASTN be set at default parameters or fasta algorithm to detect its E value is below 0.01 or 0.01.According to an embodiment preferred, the varient polynucleotide are identical with the polynucleotide of the present invention or slightly many sequences of its nucleic acid number, its probability identical with polynucleotide of the present invention is at least 99%, and using the BLASTN be set at default parameters or fasta algorithm to detect its E value is below 0.01 or 0.01.

In certain embodiments, the varient polynucleotide sequence is hybridized with described polynucleotide sequence under stringent condition." stringent condition " used herein is meant pre-washing in 6X SSC, 0.2% SDS solution; The hybridization of under 65 ℃, 6X SSC, 0.2% SDS, spending the night; In 1XSSC, 0.1% SDS, carry out twice cleaning in 65 ℃ then, each 30 minutes, and in 0.2XSSC, 0.1% SDS, carry out twice cleaning, each 30 minutes in 65 ℃.In other embodiments, the varient of described polynucleotide and/or polypeptide is separable from various mycobacteriums.In certain preferred aspects, described polypeptide variants has similar activity to described polypeptide, and this can be by for example evaluating by the ability that detects their stimulation human PBMC's cell proliferation and/or produce cytokine or IFN-hereinafter is described in detail in detail.

Polypeptide of the present invention can be connected to signal (or leading) sequence of protein N terminal, and described sequence is translated or translated back control altogether and shifts described albumen.Described polypeptide can also be connected to joint or other sequence, be easy to synthesize, purifying or identify that described polypeptide is (for example poly--as His), or to strengthen combining of described polypeptide and solid support.For example, polypeptide can be connected to immunoglobulin fc region.

Used herein is the polynucleotide that the term " x polymkeric substance " of benchmark is meant the continuous residue of the given number at least (" x ") that comprises any polynucleotide that are accredited as SEQ ID NO:5-21 with specific " x " value.The x value can be about 20 to about 600 according to concrete sequence.

Polynucleotide of the present invention comprise the polynucleotide (x-polymkeric substance) of the continuous residue of the given number at least that contains any polynucleotide of being accredited as SEQ ID NO:5-21 or its varient.According to embodiment preferred, x value preferably at least 20, more preferably at least 40, more more preferably at least 60, and most preferably at least 80.Therefore, polynucleotide of the present invention comprise and contain the polynucleotide that are accredited as SEQ ID NO:5-21 or a kind of polynucleotide of 20 polymkeric substance, 40 polymkeric substance, 60 polymkeric substance, 80 polymkeric substance, 100 polymkeric substance, 120 polymkeric substance, 150 polymkeric substance, 180 polymkeric substance, 220 polymkeric substance, 250 polymkeric substance or 300 polymkeric substance, 400 polymkeric substance, 500 polymkeric substance or 600 polymkeric substance of varient of polynucleotide wherein.

Generally speaking, polypeptide of the present invention and polynucleotide can use any preparation in numerous methods.For example, can be inserted in the expression vector by polynucleotide with coded polypeptide and in suitable host express polypeptide recombinate and produce described polypeptide.Can use in the known various expression vectors of those of ordinary skills any.Expression can be carried out in the suitable host cell with the expression vector conversion of the polynucleotide that comprise the recombinant polypeptide of encode or transfection any.Proper host cell comprises prokaryotic cell prokaryocyte, yeast cell and higher eucaryotic cells.The host cell that uses is preferably intestinal bacteria, mycobacterium, insect, yeast or mammal cell line (such as COS or CHO).To encode naturally occurring antigen, its part or its other varient of the polynucleotide of Biao Daing in this way.

Can separate polynucleotide of the present invention by following examples 1 described screening cow mycobacterium genome dna library.Perhaps, can obtain the polynucleotide of coding cow mycobacterium epi-position by the dna sequence dna of screening in suitable cow mycobacterium cDNA or genome dna library with the degenerate oligonucleotide hybridization that derives from the aminoacid sequence that separates epi-position.Can design and synthesize suitable degenerate oligonucleotide, and can be as Sambrook etc., MolecularCloning:A Laboratory Manual, CSHL Press:Cold Spring Harbor NY, 1989 describedly carry out described screening.Can use polymerase chain reaction (PCR), utilize technology well-known in the art by genomic dna or cDNA or genome dna library isolated nucleic acid probe.Can use separate probe to carry out library screening then.

Epi-position described herein has the ability of induce immune response, and irrelevant with the preparation method.More particularly, as mentioned above, described epi-position can be induced T cell, NK cell, B cell or the macrophage proliferation that derives from the mycobacterium immune body and/or be produced cytokine (for example producing IFN-and/or IL-12).

Depend on replying of needs to being used to estimate at the selection of the cell type of the immunne response of epi-position.For example, can use method preparation well-known in the art to comprise the prepared product of the T cell, NK cell, B cell and/or the scavenger cell that derive from the mycobacterium immune body, the easiest evaluation IL-12 produces.For example, can use peripheral blood lymphocytes (PBMC) prepared product, and not need further isolated cell component.For example can use density centrifugation to pass through Ficoll ^TM(Winthrop Laboratories, NY) preparation PBMC.The T cell that is used for mensuration described herein can be directly by the PBMC purifying.Perhaps, can use the enrichment T clone with anti-mycobacterium protein-active or have the T cell clone of anti-various mycobacterium protein-actives.For example, by the PBMC and the mycobacterium albumen of mycobacterium immune body are cultivated 2-4 week, can obtain described T cell clone.This makes and produces the clone of only being made up of such cell by only mycobacterium protein-specific T cell amplification.Clone these cells then, and use method well-known in the art to test itself and various albumen, to define individual T cell-specific more accurately.Can use method for example described below to measure T cell, NK cell, B cell or macrophage proliferation or generation cytokine.

In the immunogenicity epi-position, can distinguish polypeptide of the present invention and/or polynucleotide according to replying intensity and observing the individual percentage of replying in said determination with remarkable treatment characteristic.In addition, for the cell that surpasses about 25% non-branch bacillus immune body, the epi-position with remarkable treatment characteristic does not stimulate cellular proliferation or produces cytokine external, eliminates the nonspecific response because of mycobacterium response type cell thus.Therefore, the antigen of inducing T cell, NK cell, B cell or the scavenger cell prepared product of high percent mycobacterium immune body to reply (response rate to other individual cells prepared product is low) has remarkable treatment characteristic.

Evaluation with epi-position of remarkable treatment characteristic can also be carried out according to its ability of alleviating the severity of laboratory animal mycobacterium tuberculosis or other mycobacterial infections when giving as vaccine.The suitable vaccine production thing that is used for laboratory animal hereinafter is described in detail in detail.

The each several part of polypeptide of the present invention and other varient can obtain by synthetic or recombination method.Use the known technology of persons skilled in the art to produce to be less than about 100 amino acid and be less than about 50 amino acid whose synthetic polypeptide usually.For example, can use any commercially available solid phase technique, be added to the Merrifield solid-phase synthesis of the amino acid chain of growth as amino acid continuously, synthetic described polypeptide.Referring to Merrifield, J.Am.Chem.Soc.85:2149-2154,1963.Automatically the equipment of synthetic polypeptide can be to as Perkin Elmer/AppliedBioSystems, and (Foster City, supplier California) buys Inc., and can operate according to manufacturer's specification sheets.Can use the standard induced-mutation technique,, prepare natural epi-position varient as the mutagenesis of oligonucleotide fixed point specificity.Also can use the standard technique that allows preparation brachymemma polypeptide to remove the several portions of dna sequence dna.

The present invention also provides the fusion rotein that comprises first kind and second kind polypeptide of the present invention, or comprise polypeptide of the present invention and known antigen of mycobacterium tuberculosis (for example Andersen and Hansen, Infect.Immun.57:2481-2488, the 1989 38 kDa antigens of describing) fusion rotein, and the varient of described fusion rotein.In related fields, also provide the DNA that comprises first kind and second kind polynucleotide of the present invention construction.The preparation and the corresponding Recombinant Protein Expression of the construction that comprises a plurality of epi-positions of the present invention are described in detail in detail at following embodiment 4.In general, use known recombinant DNA technology to make up the polynucleotide of code book invention fusion rotein, be fitted into suitable expression vector with the dna sequence dna of described first kind and the second kind polypeptide of the present invention of will encoding respectively.With or be connected to 5 ' end of the dna sequence dna of described second peptide species of coding without will the encode 3 ' end of dna sequence dna of described first peptide species of peptide linker, make the frame of described sequence coordinate to allow mRNA that two kinds of dna sequence dnas are translated as single fusion rotein, this fusion rotein had both kept the biological activity of described first peptide species, had also kept the biological activity of described second peptide species.

Can use the peptide linker sequence, the distance that is folded into its secondary and tertiary structure with each polypeptide of sufficient to guarantee is separated described first kind and second peptide species.Use standard technique well-known in the art that such peptide linker sequence is joined in the fusion rotein.Can select suitable peptide linker sequence according to following factor: they can take flexible and extendable conformation (1); (2) they can not take to act on the secondary structure of the functional epi-position on described first kind and second peptide species; (3) lack can with the hydrophobic or charged residue of described polypeptide functional epi-position reaction.Preferred peptide linker sequence comprises Gly, Asn and Ser residue.In described peptide linker sequence, can also use other, as Thr and Ala near neutral amino acid.The aminoacid sequence that can effectively be used as joint comprises Maratea etc., Gene 40:39-46,1985; Murphy etc., Proc.Natl.Acad.Sci.USA 83:8258-8262,1986; With United States Patent (USP) the 4th, 935, No. 233 and 4,751, the aminoacid sequence of No. 180 descriptions.The length of described joint sequence can be 1 to about 50 amino acid.When described first kind and second peptide species have the nonessential-terminal amino acid district that can be used for the separation function structural domain and prevent spatial interference, do not need the peptide linker sequence.Use the known technology of persons skilled in the art, the connection dna sequence dna of encoding fusion protein is cloned in the appropriate expression system.

On the other hand, the invention provides the method for using one or more polypeptide of the present invention or fusion rotein (or polynucleotide of coding said polypeptide or fusion rotein) inducing anti-diseases in the patient (as tuberculosis) protective immunity." patient " used herein is meant any warm-blooded animal, preferably is the people.The patient can suffer from disease, perhaps can not have detectable disease or infection.In other words, can induce protective immunity prevention or treatment disease.

In related fields, can use cow mycobacterium polynucleotide of the present invention and polypeptide activated T cell and NK cell, stimulate the human PBMC to produce cytokine (particularly Th1 cytokines), produce anti-epitope antibodies and/or induce the long-term memory cell.

In order to use in described method, described polypeptide, fusion rotein or polynucleotide generally are present in medicinal compositions or the vaccine.Medicinal compositions can comprise acceptable carrier on one or more polypeptide and a kind of physiology, and wherein every kind of described polypeptide can comprise one or more above-mentioned sequences (or its varient).Vaccine can comprise one or more aforementioned polypeptides and immunologic stimulant, and all polypeptide as described are incorporated into adjuvant or liposome wherein.Such medicinal compositions and vaccine can also comprise other antigen of mycobacterium, and described other antigen joins in the fusion rotein or is present in the independent polypeptide.

On the other hand, vaccine of the present invention can contain the polynucleotide of one or more aforementioned polypeptides of encoding, but makes described polypeptide original position produce.In such vaccine, described polynucleotide may reside in any of the known various transfer systems of persons skilled in the art, comprise expression of nucleic acid system, bacterial expression system and virus expression systems.Suitable expression of nucleic acid system is included in the necessary DNA/RNA sequence (such as suitable promotor signal and terminator signal) of expression among the patient.Bacterial delivery systems comprises the bacterium (such as bacille Calmette-Guerin vaccine) that gives in the immunogenicity epi-position of the described polypeptide of its cell surface expression.In a preferred embodiment, can use virus expression systems (for example vaccinia virus or other poxvirus, retrovirus or adenovirus) to introduce described DNA and/or RNA, described system can comprise use non-virulent virus or defective type replication-competent virus.The technology that DNA and/or RNA are joined in the described expression system is well-known in the art.Described DNA can also be " naked " DNA, Ulmer etc. for example, Science 259:1745-1749, description and Cohen in 1993, Science 259:1691-1692,1993 summary.By naked DNA being coated on the biodegradable pearl that effectively is transported in the cell, can increase the absorption of naked DNA.The method that comprises the polynucleotide sequence of DNA and/or RNA is included in United States Patent (USP) the 5th, 580, and No. 859 and 5,589, disclosed method in No. 466.

Aforesaid polynucleotide vaccine can with polypeptide of the present invention or known antigen of mycobacterium (as above-mentioned 38kDa antigen) simultaneously or sequential giving.For example, can behind the DNA that gives code book invention polypeptide, give antigen, so that strengthen the protective immunity effect of described vaccine.

Approach that gives and frequency and give dosage and change to some extent between individuality can be identical with those conditions of using in the present Mycobacterium bovis BCG immunity.Generally speaking, described medicinal compositions and vaccine can be by (for example by sucking) or orally gives in injection (for example intracutaneous, intramuscular, intravenously or subcutaneous), the nose.Can give 1 to 3 dose in week at 1-36.Preferably gave 3 doses at interval, after this can regularly carry out the booster immunization inoculation with 3-4 month.Can take alternatives for individual patient.Proper dosage is when giving as mentioned above, and the amount of polypeptide or polynucleotide can excite the patient to produce is enough to protect described patient to avoid the mycobacterial infections immunne response of 1-2 at least.In general, the polypeptide amount that potion contains (or polypeptide amount of potion polynucleotide original position generation) is the about 100mg of the about 1pg-of every kg host, is generally the about 1mg of the about 10pg-of every kg host, is preferably the about 1 μ g of the about 100pg-of every kg host.Suitable dosage ranges will change to some extent according to described patient's size, but be generally the about 5ml of about 0.1ml-.

Although can use the known arbitrary suitable carrier of persons skilled in the art in medicinal compositions of the present invention, the type of carrier will change to some extent according to giving mode.Give for parenteral, such as subcutaneous injection, described carrier preferably includes water, salt solution, alcohol, fat, wax or damping fluid.For orally give, can use any or solid carrier in the above carrier, as N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, talcum powder, Mierocrystalline cellulose, glucose, sucrose and magnesiumcarbonate.Can also use the carrier of biodegradable microballoon (for example poly(lactic acid) galactoside) as medicinal compositions of the present invention.Suitable biodegradable microballoon for example is disclosed in United States Patent (USP) the 4th, 897, No. 268 and the 5th, 075, No. 109.

In vaccine of the present invention, can use in the various immunologic stimulants any,, reply with non-specific enhancing immunity such as adjuvant.Most of adjuvants comprise the nonspecific stimulation agent (such as the cow mycobacterium of lipoid A, bordetella pertussis (Bordetella pertussis), mycobacterium tuberculosis or following argumentation) that is used for avoiding quick catabolic material of antigen (such as aluminium hydroxide or mineral oil) and immunne response.Suitable adjuvant is commercially available, as Freund's incomplete adjuvant and Freund's complete adjuvant (Difco Laboratories, Detroit, MI), and the Merck adjuvant 65 (Merck and Company, Inc., Rahway, NJ).Other suitable adjuvant comprises alum, biodegradable microballoon, monophosphoryl lipid A and Quil A.

It has been illustration that following embodiment is provided, rather than provides constraints.

Embodiment 1

Clone and selection immunogenicity cow mycobacterium epi-position

In 37 ℃ at substratum 90 (yeast extract, 2.5g/l; Tryptones, 5g/l; Glucose, 1g/l) middle cow mycobacterium (ATCC preserving number 15483, Rockville, MD) 4 days of cultivating., producing under the segmental condition of about 0.25kbDNA with restriction endonuclease Sau3A digestion then by these cellular segregation genomic dnas according to standard method.Use QIAquickPCR purification system (Qiagen, Venlo, The Netherlands) the described fragment of purifying.

For expressing the clone cow mycobacterium DNA of three kinds of different frames, by insert human growth hormone signal peptide (to promote the recombinant protein secretion) modification pcDNA3 expression vector with three kinds of difference 3 ' primer amplifications (Invitrogen, Carlsbad, CA).These primers allow 1 or 2 extra base pairs are inserted in the PCR product, to move the frame of expressed polypeptide.Described primer is AD105 (human growth hormone 5 ' primer; SEQ ID NO:1) and three-type-person's tethelin (hGH) 3 ' primer AD106, AD107 and AD108 (being respectively SEQ ID NO:2-4).By digesting by most of hGH sequence of removing leader sequence cleavage site downstream in these PCR fragments with restriction endonuclease BsgII.Then hGH PCR fragment cloning is gone in the pcDNA3 expression vector, then with restriction endonuclease HindIII and BamHI digestion.SEQ ID NO:5-7 has provided and has inserted segmental nucleotide sequence, and SEQ ID NO:61-63 provide amino acid sequence corresponding respectively.Go into the BamHI cloning site of chimeric pcDNA3/ human growth hormone carrier (pcDNA3-hGH1 ', pcDNA3-hGH2 ' and pcDNA3-hGH3 ') by the 0.25kb cow mycobacterium PCR fragment cloning that will prepare as mentioned above, make up three kinds of expression libraries (each libraries of three frames).The bacterial colony that transforms with described library construction thing prepares photo etching (replica lift master plate) and storage.To be divided into 500 storehouses by the plasmid DNA of these bacterium colony preparations, 40-50 plasmid DNA contained in every storehouse.Use lipofection amine reagent (BRL Life Technologies, Gaithersburg MD) described DNA is transfected in the COS7 cell, and utilize splenocyte measure to detect the immunogenicity characteristic of every group of product, wherein as described belowly measure hot deactivation cow mycobacterium with ELISA and excite the immune mouse spleen cell culture to produce IFN-γ first.

To excite the recombinant polypeptide coding plasmid storehouse of immunne response (to increase the ability mensuration that IFN-γ produces in the splenocyte mensuration) to be subdivided into littler storehouse again, 10 plasmids are contained in each storehouse, these storehouses are transfected in the COS7 cell again.As mentioned above the culture supernatant of these cells being carried out splenocyte measures.

After the three-wheel screening, identify the plasmid of 120 coding recombinant polypeptides, described recombinant polypeptide irritation fever deactivation cow mycobacterium immune mouse spleen cell produces IFN-γ.Use other two kinds of 120 COS7 cell culture supernatants measuring screening with these plasmid transfections, described two mensuration are that mouse memory is measured and human peripheral blood mononuclear cell (PBMC) measures.In the mouse long-term memory is measured, with 10 of sublethal dose ⁴Colony-forming unit (CFU) mycobacterium tuberculosis injection mouse.After 4 weeks, treating 4 weeks of mouse again with microbiotic, to cure the m tuberculosis infection of mouse, then is the stationary phase in 4 weeks.Inject for the second time slip-knot nuclear mycobacterium (5 * 10 ⁵CFU) after 4 days, use above-mentioned splenocyte to measure the immunogenicity that detects the plasmid product.

In PBMC measures, with its 120 plasmid transfection COS7 cell culture supernatants of ability screening of inducing mycobacterium immunity people donor peripheral blood cells T cell proliferation and producing IFN-γ.Known these donors are PPD (purified protein derivative of the mycobacterium tuberculosis) positive, confirm that its T cell propagation takes place under the PPD effect and produces IFN-γ.In the substratum that contains the RPMI 1640 that adds 10% (v/v) autoserum, penicillin (60 μ g/ml), Streptomycin sulphate (100 μ g/ml) and glutamine (2mM), cultivate donor PBMC and COS7 culture supernatant.After 3 days, by taking out 50 μ l substratum, mensuration IFN-γ level as described below in every hole.Culture plate is 4 days again, uses the tritiate thymidine burst process 18 hours in 1 μ Ci/ hole then, and harvested cell also uses scintillometer to measure tritium and takes in.The level of measuring moderate stimulations propagation at two duplications is judged to the positive than the supernatant liquor of the high twice of propagation that observes at single culture base culturing cell.

Following use enzyme-linked immunosorbent assay (ELISA) detects IFN-γ.By (MA) phosphate-buffered saline (PBS) solution makes described antibody sandwich elisa plate in 4 ℃ of incubations 4 hours for Endogen, Wobural at the mouse monoclonal antibody of people IFN-γ with each hole and 1 μ g/ml.At room temperature sealed each hole 1 hour with the PBS that contains 0.2%Tween 20.In PBS/0.2%Tween 20, clean described plate 4 times then, and in elisa plate with 1: 2 dilute sample of substratum, under room temperature, be incubated overnight.Clean described plate again, in every hole, be added in the anti-people IFN-of the biotinylation multi-clone rabbit γ serum (Endogen) that is diluted to 1 μ g/ml among the PBS.The described plate of incubation 1 hour under room temperature then cleans and is added among the PBS with 1: 4, and the avidin A of the coupling horseradish peroxidase of 000 dilution (Vector Laboratories, Burlingame, CA).In the room temperature incubation after 1 hour, clean plate also adds O-Phenylene Diamine (OPD) substrate again.After 10 minutes with 10% (v/v) HCl termination reaction.Detect the optical density(OD) (OD) of 490nm.The culture supernatant that OD is higher than the average OD twice of single culture base culturing cell in two duplications are measured is judged to the positive.

Result according to these two mensuration identifies 59 plasmids that coding contains the recombinant polypeptide of immunogenicity determinant or epi-position.Find that these epi-positions excite mouse and human immune, and with induce the mycobacterium tuberculosis immunogenicity determinant cross reaction of replying for a long time.Test these plasmids and in following tuberculosis mouse model, induced the ability of protective immunity.At the tibialis anterior muscle IM of anesthetized mice injection every kind of plasmid (100 μ g DNA), 2 week injections are three times at interval.After 9 weeks, with mycobacterium tuberculosis (5 * 10 ⁵CFU) attack mouse.The organ homogenate for preparing lung and spleen in the 12nd week, and adding the 7H9 substratum upper flat plate cultivation of oleic acid-albumin dextrose catalase (OADC), to measure the CFU number that every kind of homogenate exists.Two week of incubation back record result.

Use method described above to select 8 plasmids that contain the immunogenicity epi-position.Identify the open reading-frame (ORF) (ORF) of inferring in these constructions after, the cow mycobacterium fragment subclone that will only contain the ORF part is as mentioned above gone among the pcDNA3-hGH '.These plasmids are called DNA5, DNA9, DNA26, DNA27, DNA29, DNA37, DNA44 and DNA45.In DNA9, identify three kinds of ORF that are called A, B and C.Open reading-frame (ORF) B and C are oppositely, and be discarded.Clone ORF A separately, the plasmid of acquisition is called DNA9A.SEQ ID NO:13-21 has provided measured DNA5, DNA9A, DNA26, DNA27, DNA29, DNA37, DNA42, DNA44 and DNA45 respectively and has inserted segmental genomic dna sequence, and SEQ ID NO:69-77 provides the expection aminoacid sequence of corresponding ORF respectively.In the insertion fragment of plasmid DNA 5 and DNA27, identify more than one epi-position.These epi-positions can not be separated by the clone, and test is multi-epitope in all mensuration.SEQ IDNO:8-12 has provided the epi-position 1 of epi-position 1, epi-position 2 and epi-position 3 and DNA27 of DNA5 and the mensuration genomic dna sequence of epi-position 2 respectively, and SEQ ID NO:64-68 provides corresponding expection aminoacid sequence respectively.Use the epi-position dna sequence dna of FASTA computerized algorithm comparative determination and the sequence in the EMBL DNA database.Use the sequence in the corresponding expection of FASTX computerized algorithm contrast protein sequence (albumen of the DNA translation in each of 6 frames) and the SwissProt database.Dna sequence dna that comparison on March 21st, 1999 SEQ IDNO:8-12 provides and the sequence in the EMBL DNA database (using FASTA), and compare aminoacid sequence and the sequence in the SwissProt database (using FASTX) that SEQ ID NO:64-77 provides.

Use FASTX to measure discovery as mentioned above, the identity of the sequence in the expection aminoacid sequence of DNA5 epi-position 2, DNA27 epi-position 1, DNA9A, DNA29, DNA37, DNA44 and DNA45 (being provided by SEQ ID NO:65,67,70,73,74,76 and 77 respectively) and the SwissProt database is less than 50%.Use FASTX to measure discovery as mentioned above, the identity of the sequence in DNA5 epi- position 1 and 3 expection aminoacid sequence (being provided by SEQ ID NO:64 and 66 respectively) and the SwissProt database is less than 75%.Sequence with expection aminoacid sequence (providing by SEQ ID NO:68 and the 71 respectively) coupling of DNA27 epi-position 2 and DNA26 is provided.Following table 1 has shown the result who uses the sequence in FASTX aminoacid sequence more of the present invention and the SwissProt database as mentioned above, and wherein " identical residue number " is the identical residue number of contrast part.

Table 1

	?SEQ?ID ?NO：	Length (residue)	Correlation length (residue)	The identical residue number	Identical residue percentage %
						DNA5, epi-position 1	????64	????13	????11	????8	????61
DNA5, epi-position 2	????65	????32	????31	????10	????32
						DNA5, epi-position 3	????66	????26	????25	????14	????53
?DNA9A	????70	????75	????68	????24	????32
						?DNA26	????71	????97	Miss
DNA27, epi-position 1	????67	????38	????30	????16	????42
						DNA27, epi-position 2	????68	????11	Miss
?DNA29	????73	????46	????40	????17	????37
						?DNA37	????74	????87	????80	????26	????32
?DNA44	????76	????44	????35	????17	????38
						?DNA45	????77	????59	????52	????21	????35

Embodiment 2

Express recombinant epi-position in prokaryotic cell prokaryocyte and eukaryotic cell

Epi-position DNA subclone is gone in the carrier, with express polypeptide in bacterium and eukaryotic cell.Bacterial expression vector be modified form pET16 carrier (Novagen, Madison, WI).Except DNA9, the insertion fragment of all plasmids is all used primer AD136 and AD133 (being respectively SEQ ID NO:22 and 23) amplification, and connect into EcoRI digestion and with archaeal dna polymerase PfuI (Stratagene by flush end, La Jolla CA) clones in the pET16 carrier of end-filling.The insertion fragment of DNA9A increases with AD250 and AD251 (being respectively SEQ IDNO:24 and 25), and is cloned into as mentioned above in the pET16 carrier.

For in eukaryotic cell, expressing described polypeptide, modify pcDNA3 carrier (Invitrogen), comprise a histidine mark with 3 ' end at cloning site.This can be by finishing among the pcDNA3 that double chain oligonucleotide AD180/AD181 is cloned into BamHI and EcoRI digestion.SEQ ID NO:26 and 27 has provided the sequence of oligonucleotide AD180 and AD181 respectively.Use pcDNA3-hGH ' construction as dna profiling, insert fragment with hGH specificity N end 5 ' primer AD134 (SEQ ID NO:28) and epitope specificity 3 ' terminal primer amplification plasmid.SEQ ID NO:29-37 has listed the sequence of epitope specificity 3 ' primer AD151 (DNA5), AD153 (DNA26), AD154 (DNA27), AD155 (DNA29), AD158 (DNA42), AD159 (DNA44), AD160 (DNA45), AD167 (DNA37) and AD182 (DNA9) respectively.

Modify this carrier once more,, make the hGH ' sequence in this construction be reduced to preceding 5-terminal amino acids of only surplus leader sequence and hGH sequence to remove the unnecessary sequence (42 Nucleotide) between hGH leader sequence and the expressed sequence.Use pcDNA3-hGH3 ' construction as template, use primer AD105 (SEQ ID NO:1) and AD222 (SEQ ID NO:38) to shorten fusion partner through pcr amplification.Be cloned into pcDNA3-His at HindIII and BamHI site, the construction of acquisition is called pcDNA3-hGHls/His.SEQ ID NO:39 has provided the mensuration dna sequence dna of the hGH-fusion partner that is cloned among the pcDNA3-hGH-ls, and corresponding amino acid sequence is shown in SEQ ID NO:78.Use primer AD233 and AD226 (being respectively SEQ ID NO:40 and 41) to insert the construction that fragment is formed by DNA9A by the pcr amplification preparation.

Embodiment 3

The immunogenicity of recombination epitope construction

Present embodiment has been described the result that the recombination epitope with the DNA of 8 selections or recombinant polypeptide form carries out immunogenicity research.A. stimulated in vitro human peripheral blood mononuclear cell (PBMC) breeds and secretion IFN-(IFN-γ)

The recombination epitope (1 and 10 μ g) that the pET16 bacterial expression system is expressed and human PBMC are in 37 ℃ of cultivations.After 48 hours, detect IFN-γ secretion with enzyme-linked immunoassay (ELISA) according to standard method.With the parallel culture of tritiate thymidine pulse, and use the synthetic PBMC of the evaluation propagation of DNA.For relatively, also cell is cultivated with mycobacterium tuberculosis purified protein derivative (PPD), and described cell is cultivated as negative control with PBS.

As shown in table 2, all recombination epitopes can both stimulate some PBMC sample generation IFN-γ at least.In 12 PBMC samples being tested, 10 samples are the PPD positive, and promptly the PBMC of these samples produces IFN-γ when cultivating with PPD, and 2 samples are the PPD feminine gender.Think that the PBMC reaction is positive during high 3 times of the IFN-γ that produces than PBS control sample at least when the IFN-γ amount that produces.Recombination epitope 5 (corresponding to DNA5) and 44 (corresponding to DNA44) stimulate 100% PPD ⁺Sample produces IFN-γ.Recombination epitope 27 (corresponding to DNA27) stimulates 80% PPD ⁺Sample produces IFN-γ.Recombination epitope 26 and 37 (corresponding respectively to DNA26 and DNA37) stimulates 70% PPD ⁺Sample produces IFN-γ, and epi-position 45 (corresponding to DNA45) stimulates 20% PPD ⁺The PBMC sample.PPD ^-The PBMC sample does not all have significant reaction to any recombination epitope.This proves that described epi-position has immunogenicity in human body, and triggers the anamnestic reaction of the donor sample that contacts mycobacterium in advance.

Table 2

Recombination epitope stimulates the human PBMC to produce IFN-γ

The human PBMC	The PBS contrast	The PPD contrast	Recombination epitope
									??26	??37	??44	??45	??5	??27
			?G97022	＜0.1	??0.16	??0.3	??0.3	??0.4							??＜0.1	??0.5	??0.1
?G97037	＜0.1	??0.3							??0.3	??0.3	??0.8	??0.2	??0.3	??0.3
			?G97001	＜0.1	??4.5	??0.6	??1	??3.5							??0.2	??1.8	??1.7
?G97008	＜0.1	??4.4							??0.16	??0.5	??4	??＜0.1	??0.73	??0.9
			?G97011	0.25	??4.9	??0.9	??0.5	??1.2							??0.25	??2.8	??1.2
?G97030	＜0.1	??1.8							??0.5	??0.2	??3.5	??0.1	??1.8	??3
			?G97033	0.12	??4.5	??0.5	??0.25	??3.4							??0.2	??1.7	??1
?G97010	＜0.1	??＞4							??＞4	??＞4	??＞4	??1	??＞4	??＞4
			?G97028	＜0.1	??＞4	??＞4	??＞4	??＞4							??1.2	??＞4	??＞4
?G97020	＜0.1	??1							??0.3	??0.25	??＞4	??＜0.1	??1.5	??0.5
			?G97032	＜0.1	??＞4	??0.5	??1.2	??＞4							??＜0.1	??1.4	??0.5
?G97035	＜0.1	??3.5							??0.4	??3.5	??＞4	??＜0.1	??1	??1

^*The result represents with ng IFN-γ/ml.

The two proliferative response has further proved the immunogenicity of described epi-position to the people to PPD and recombination epitope by human PBMC's sample.Represent that with stimulation index described recombination epitope stimulates the ability of PBMC propagation.When the propagation stimulator is bred high 5 times than the average background that only has the substratum contrast to produce, then think and breed the stimulator positive.As shown in table 3, find that all recombination epitopes all have the stimulation ability of the PBMC propagation of some human PBMC's sample at least.Recombination epitope 26 and 27 stimulates the PBMC propagation of 92% described sample, and the described sample stimulus propagation of 5,37 and 44 pairs 83% of recombination epitopes.The described sample stimulus PBMC propagation that epi-position is 45 pairs 17%.PPD is 83% to the hormesis of PBMC.

Table 3

Hormesis to PBMC propagation

The human PBMC	Recombination epitope
								???PPD	????5	????26	????27	????37	????44	????45
	?G97022	????5 *	????12	????14	????3	????20	????20								????1
?G97037								????35	????12	????25	????20	????15	????30	????1
	?G97001	????5	????6	????6	????5	????8	????7								????1
?G97008								????60	????20	????30	????15	????40	????60	????2
	?G97011	????40	????33	????30	????27	????30	????30								????3
?G97030								????100	????140	????120	????140	????120	????140	????30
	?G97033	????20	????12	????12	????11	????10	????15								????1
?G97010								????2	????4	????2	????6	????2	????4	????2
	?G97028	????60	????48	????60	????40	????55	????52								????12
?G97020								????10	????10	????10	????8	????10	????10	????1
	?G97032	????45	????30	????35	????33	????38	????42								????2
?G97035								????3	????4	????5	????6	????3	????4	????2

^*Represent the result that the human PBMC breeds with stimulation index.B. use DNA epi-position immune mouse

After the coding DNA of 8 recombination epitopes injecting pcDNA-hGH '/His or pcDNA3-hGHls/His construction, in the BALB/cByJ mouse, induce the protective immunity of anti-m tuberculosis infection subsequently.

Observe protective immunity after subsequently with m tuberculosis infection and induce,, when lung homogenate CFU on average descends 0.5log, think that then the inductive protective immunity is positive when comparing with the average CFU of immune control mice not.Do not insert segmental plasmid and be used as contrast.Also the immunogenicity of the minimizing of the CFU behind the epi-position dna immunization with known Mycobacterium bovis BCG compared.The result is clear to be shown, the CFU of all test mouse all reduces, and points out described recombination epitope DNA to induce protective immunity.In 6 groups, CFU descends and descends suitable above the CFU decline of 50%, 3 group and injection Mycobacterium bovis BCG inductive CFU.

Table 4 is by exempting from 8 pcDNA-hGH '/His or the heredity of pcDNA3-hGHls/His construction

The epidemic disease mouse is induced protective immunity

The epi-position construction	SEQ?ID?NO：	The mouse number	The mouse number that CFU descends	The mouse percentage % that CFU descends
					Control plasmid		????14	????2	????14
??DNA5	????13	????11	????4	????36
					??DNA9	????14	????13	????7	????55
??DNA26	????15	????10	????7	????70
					??DNA27	????16	????13	????7	????55
??DNA29	????17	????13	????7	????55
					??DNA37	????18	????13	????8	????62
??DNA44	????20	????13	????6	????46
					??DNA45	????21	????13	????8	????62
Mycobacterium bovis BCG		????23	????17	????74

Following evaluation is inductive cytotoxic T lymphocyte (CTL), cytokine (IFN-γ, IL-4, IL-6 and IL-10) and propagation and antibody response during with 8 pcDNA-hGH '/His or pcDNA3-hGHls/His construction inherited immunity.CTL measures:

(Manassas VA), detects molten cell (CTL) activity of dna immunization BALB/cByJ mouse boosting cell according to the standard two-step approach for ATCC preserving number CRL-163, American type culture collection to use MHC haplotype coupling target cell BALB-3T3.By with 8 pcDNA-hGH '/His or pcDNA3-hGHls/His epi-position DNA construction transfection BALB/3T3 cells, prepare target cell.Select (G418 by Geneticin; Gibco BRLLife Technologies) produce stably transfected cell line, and by the limiting dilution isolated mononuclear cell.Use primer AD105 and AD181 (being respectively SEQ ID NO:1 and 27) to select to express the clone of epi-position by RT-PCR.In the presence of mitomycin treatments B ALB-3T3 cell, in the cDMEM that replenishes 10%FCS, cultivate the dna immunization mouse boosting cell, with the external toxicity T of irritation cell again cell with coupling epi-position DNA transfection.In 37 ℃ at 10%CO ₂Following incubation culture 6 days.A part and usefulness by every kind of irritation cell culture of incubation ⁵¹The DNA coupling target cell of Cr pulse discharges in the detection cell culture supernatant liquid ⁵¹Cr, thereby monitoring dissolved cell activity.As shown in table 5, the splenocyte that detects 4 kinds of test dna construction immune mouses has the specific CTL activity.Cytokine and proliferative response:

Producing and proliferative response with the external cytokine of postevaluation dna immunization BALB/cByJ mouse boosting cell that stimulates again of recombination epitope.Respectively as mentioned above by ELISA and 3H-thymidine pulse detection cytokine and proliferative response.As shown in table 5, the splenocyte of 6 test group produces Th1 cytokine IFN-γ.In the supernatant liquor of irritation cell, do not detect Th2 cytokine (for example IL-4, IL-6 and IL-10).Proliferative response is low, and only detects in two immune group mouse boosting cells.Antibody response

2 weeks were collected the blood sample of three kinds of dna immunization BALB/cByJ mouse after the last DNA of injection, and prepared serum according to standard method.Whether detect anti-epitope antibodies with ELISA exists.With 500ng recombination epitope bag by each hole of microtiter plate.Join in each hole by serum and to detect antibody titers serial dilution.The ELISA method detects antibody titers as described above.As shown in table 5, in two blood samples of test, detect anti-epitope antibodies.Also use standard method to carry out ELISA and detect, belong to the IgG1 hypotype or belong to the IgG2a hypotype to measure described antibody.The result shows that described antibody belongs to the IgG2a hypotype, and the IgG2a hypotype is the feature of Th1 antibody response.

The data of general introduction show in table 5, with epi-position dna immunization induce immune response in mouse.And what detected cell response and humoral response showed generation in described dna immunization mouse is that the Th1 type is replied.

Table 5

By exempting from 8 pcDNA-hGH '/His or the heredity of pcDNA3-hGHls/His construction

Epidemic disease mouse inductive cytotoxic lymphocyte is induced, cytokine is reacted,

Propagation and antibody response

The epi-position construction	????SE ????ID ????NO：	CTL induces (specificity cracking %) *	The cytokine reaction (IFN-γ, ng/ml) **	Proliferative response (stimulation index)	Antibody (titre)
						??DNA5	????13	??30	????15	Do not detect	Do not detect
??DNA9A	????14	??10	????NT ***	??NT	????NT
						??DNA26	????15	Do not detect	????30	??7	Do not detect
??DNA27	????16	Do not detect	????33	??3	????1/100
						??DNA29	????17	Do not detect	????NT	??NT	????NT
??DNA37	????18	Do not detect	????18	Do not detect	Do not detect
						??DNA44	????20	??25	????23	Do not detect	????1/100
??DNA45	????21	??20	????12	Do not detect	Do not detect

^*Data presented is the cracking average percentage of three mouse boosting cells.Deducted the non-of control cells

The specificity cracking. ^*Data presented is the average IFN-by three parts of spleen cell cultures things acquisitions of three mouse

γ(ng/ml)。The background IFN-γ that control cells produces is 5-7ng/ml. ^{* *}NT=does not test.

Embodiment 4

Clone's strategy of cow mycobacterium multi-epitope construction

8 epi-positions that assembling embodiment 4 measures are to form the multi-epitope construction.Specifically, with the described DNA of primer amplification that contains BglII 5 '-extension and BamHI 3 '-extension, subsequently it is cloned into the BamHI site of pcDNA3/hGHls/His.Primer is AD223, AD226, AD229, AD230, AD231, AD232, AD233, AD234, AD235, AD236, AD256, AD258, AD259, AD260, AD261 and AD262 (being respectively SEQ ID NO:40-55).

At first the insertion fragment cloning of plasmid DNA 9A is gone into the BamHI site of pcDNA3-hGHls/His.Only in the BamHI site of clone's joint 3 ' described carrier of terminal reconstruct, and with beyond the DNA5 all other insert that fragments are sequential to be cloned into identical site.Connect by flush end at last the insertion fragment cloning of plasmid DNA 5 is gone into the end-filling BamHI site of pcDNA3/hGHls/His.After this step, the epi-position of various combinations is cloned into the pcDNA3-hGHls/His carrier.SEQ ID NO:56-58 has shown the mensuration dna sequence dna of three kinds of multi-epitope constructions (being called ME/A, ME/B and ME/D) of being made up of 8 aggressiveness multi-epitopes respectively, and SEQ ID NO:79-81 has shown the corresponding aminoacid sequence of expection respectively.In these multi-epitope constructions each all comprises each in described 8 epi-positions, but order is different.

In order in bacterium, to express the multi-epitope recombinant protein, the insertion fragment subclone of plasmid ME/A, ME/B and ME/D is gone into modified form expression vector pET16.The combination of all 8 aggressiveness Polyepitope DNAs all respectively its 5 ' and 3 ' end have DNA9A and DNA5.Use primer AD272 and AD273 (being respectively SEQ ID NO:59 and 60) amplification plasmid to insert fragment, and the amplified fragments that connects purifying by flush end is cloned into EcoRI digestion and mend flat terminal pET16 carrier with archaeal dna polymerase PfuI (Stratagene).Use the e. coli host cell express recombinant protein and use the standard method purifying according to manufacturer's method.

Embodiment 5

With cow mycobacterium multi-epitope construction immune mouse

Present embodiment is illustrated the protective immunity to its anti-m tuberculosis infection subsequently behind the multi-epitope construction of BALB/cByJ injected in mice DNA or recombinant polypeptide form.

The BALB/cByJ mouse is divided into three groups, 6 every group, accepts different treatment.The 1st group of mouse is with 1 dose of 500 μ g Mycobacterium bovis BCG or the immunity of PBS intraperitoneal.The 2nd group of mouse with 100 μ g ME/D DNA or empty carrier DNA with the interval in three weeks mouse tibialis anterior muscle intramuscular immunity three times.The 3rd group mouse with 1 or 2 dose 50 μ g in IFA reorganization ME/D or the reference protein of 50 μ g in IFA with the interval intraperitoneal immunity in three weeks.The reference protein GV14B that exists albumen to form by non-natural derives from the coding DNA of the cow mycobacterium homologue of mycobacterium elongation factor G, and it is gone among the pET16g3 with reverse cloning.

In last immunity three weeks of back, examine mycobacterium (5 * 10 with slip-knot ⁵CFU) attack mouse.The organ homogenate of preparation lung and spleen after three weeks, and adding the 7H9 substratum upper flat plate cultivation of oleic acid-albumin dextrose catalase (OADC), to measure the CFU number that exists in every kind of homogenate.Two week of incubation back record result.

Observe protective immunity after subsequently with m tuberculosis infection and induce,, when lung and spleen homogenate CFU on average descend 0.5log, think that then the inductive protective immunity is positive when comparing with the average CFU of immune control mice not.CFU behind comparison ME/D DNA or the reorganization ME/D polypeptide immune reduces the immunogenicity with known Mycobacterium bovis BCG.Result (Fig. 1) shows that the ME/D DNA and the lung of reorganization ME/D construction immune mouse and the CFU of spleen reduce.The decline of lung and the two CFU of spleen is higher than with the lung CFU decline (table 4) after the single epi-position construction immunity immunity, and this has proved the protective immunity of ME/D DNA and rME/D polypeptide.

Embodiment 6

The immunogenicity of cow mycobacterium multi-epitope construction

Present embodiment has been described the result that the multi-epitope construction with 3 kinds of DNA or recombinant polypeptide form carries out immunogenicity research.A. recombinant multi-epitope construction ME/A, ME/B and ME/D stimulate mouse lymph nodal cell in-vitro multiplication and secretion of gamma-IFN

According to the T cell proliferation of its inducing mouse lymph-node cell and ability screening recombinant multi-epitope construction rME/A, rME/B and the rME/D of IFN-γ.Measure recombinant multi-epitope construction rME/A, rME/B and the subcutaneous immune BALB/cByJ mouse of rME/C of diluting with equal-volume IFA with 10 μ g at each palmula in order to carry out this.Control group mice is received in the PBS among the IFA.Put to death mouse after 9 days, take out lymphoglandula.In the presence of 0,0.125,0.25 or 0.5 μ M rME/A, rME/B or rME/D and reference protein GV14B, in the substratum that contains the DMEM that adds 10% (v/v) autoserum, penicillin (60 μ g/ml), Streptomycin sulphate (100 μ g/ml) and glutamine (2mM), cultivate lymph-node cell.After 3 days,, measure IFN-γ level as mentioned above by taking out 50 μ l substratum in every hole.Cultivated again described dull and stereotyped 4 days, and used the tritiate thymidine pulse 18 hours in 1 μ Ci/ hole then.Harvested cell also uses scintillometer to measure the tritium picked-up.The level that to measure moderate stimulations propagation at two duplications is judged to the positive than the supernatant liquor of the high twice of the observed propagation of single culture base culturing cell.

Fig. 2 A-D has shown the result of mouse proliferation experiment.All the three kinds of equal induction of immunity mouse lymph of recombinant multi-epitope construction nodal cells proliferative responses.Three kinds of recombinant multi-epitope construction rME/A, rME/B are similar with rME/D inductive propagation level (being respectively Fig. 2 A, B and C), show that described construction antigenicity is identical.Proliferative response (Fig. 2 D) does not appear in the control mice of PBS immunity.

Fig. 3 A-C has shown stimulation rME/A, rME/B or rME/D immune mouse lymph-node cell excretory IFN-γ level respectively.All three kinds of recombinant multi-epitope constructions all stimulate the lymph-node cell secretion of gamma-IFN, the irritation level of rME/B the highest (Fig. 3 B).PBS stimulated control mouse cell secretion can not detection limit IFN-γ (Fig. 3 D).These results show, induce the Th1 immunne response with the immunity of described multi-epitope construction in mouse.B. the lymph-node cell of recombinant multi-epitope construction ME/D and ME/D DNA stimulated in vitro different approaches immune mouse and splenocyte are bred and secretion of gamma-IFN

In these experiments, with described recombinant multi-epitope construction rME/D or its dna form is subcutaneous, intraperitoneal or intramuscular immune mouse, stimulate the lymph-node cell of mouse or splenocyte with inducing T cell propagation and generation IFN-γ.Perhaps with the 10 μ g rME/Ds of potion in IFA at each palmula subcutaneous or with potion in IFA 50 μ g rME/D intraperitoneal or with three dose of 100 μ g ME/D DNA with the immune BALB/cByJ mouse of three weekly interval intramusculars.Control mice uses PBS through described three kinds of different immunization route immunity.Put to death after 9 days through subcutaneous and intraperitoneal approach mice immunized, take out lymph-node cell (subcutaneous immunity) or splenocyte (intraperitoneal immunity).The splenocyte of immune mouse was gathered in the crops in the intramuscularly immunity in back 15 days the last time.Measure the propagation of these cells and the IFN-γ of generation as mentioned above.

Fig. 4 and Fig. 5 have shown these result of experiment.In Fig. 4 A, rME/D or the lymph-node cell of ME/D dna immunization mouse and the specificity multiplication reaction of splenocyte have been shown.By contrast, Fig. 4 B has shown the low propagation of control mice.Equally, stimulate lymph-node cell and the splenocyte secretion of gamma-IFN (Fig. 5 A) of rME/D or ME/D dna immunization mouse, and the lymph-node cell and the splenocyte of PBS immunity control mice are secreted low-level IFN-γ.C. the single epi-position stimulated in vitro recombinant multi-epitope construction rME/D of multi-epitope construction or ME/D DNA are through lymph-node cell and the splenocyte propagation and the secretion of gamma-IFN of different approaches immune mouse

In these experiments, stimulate lymph-node cell and the splenocyte of the multi-epitope construction ME/D of recombinant multi-epitope construction rME/D or dna form again through different immunization route immune mouses with single epi-position DNA5, DNA9, DNA26, DNA27, DNA29, DNA37, DNA44 and the DNA45 of recombinant forms.Experimental procedure is identical with above general introduction.These result of experiment are shown in Fig. 6 A-B and Fig. 7 A-B.Detecting specificity multiplication reaction and IFN-γ secretion (Fig. 6 A and Fig. 7 A) in the stimulated cells again with epi-position DNA5, DNA9A, DNA26, DNA37 and DNA44.Epi-position DNA27, DNA29 and DNA45 show propagation and IFN-γ secretion at least one immune group.In the cell of PBS immunity control mice, observe low-level propagation and IFN-γ generation (Fig. 6 B and Fig. 7 B).Data show when described epi-position is offered to immunity system as the immunogenic integral part of polycomponent all have antigenicity separately.D. cytokine produces

Mensuration as described below is with the multi-epitope construction ME/D of dna form immunity and the cytokine that produces with the mouse boosting cell that rME/A or rME/D stimulate again.With three dose of 100 μ gME/D DNA interval intramuscular immunity BALB/cByJ mouse with three weeks.Back 15 days of last injection is put to death mouse and is taken out splenocyte.Stimulate splenocyte again with rME/A, rME/D or reference protein GV14B, and produce the supernatant liquor of cytokine according to the standard method screening.Table 6 has provided the situation of splenocyte generation cytokine.

Table 6

The BALB/cByJ mouse boosting cell excretory cytokine of ME dna immunization

	???????????IL-2 *			????????????IL-4 *			???????????IL-6 *
										Plasmid	??rME/A	?rME/D	?GV14B	?rME/A	?rME/D	?GV14B	?rME/A	?rME/D	?GV14B
?ME/A	??122	??125	??＜50	??42	??56	??39	??147	??196	??＜30
										?ME/B	??267	??200	??＜50	??20	??＜10	??＜10	??79	??56	??＜30
?ME/D	??96	??77	??＜50	??13	??＜10	??＜10	??131	??98	??＜30
										Contrast	??＜50	??＜50	??＜50	??＜10	??＜10	??＜10	??＜30	??＜30	??＜30

^*Detect cytokine concentration, standard error＜10% with pg/ml

As shown in table 6, rME/A and rME/D stimulate splenocyte secretion IL-2 and the IL-6 of mouse.IL-2 is an excretory cytokine during the Th1 type immunne response, and this provides further evidence to show that described multi-epitope construction excites Th1 type immunne response.Cytokine IL-6 at mouse immune lungy, play an important role (Ladel etc., Infec.Imm.65:4883-4849,1997).After testing, do not secrete Th2 cytokines IL-4.E. antibody produces

Two weeks of DNA injection back are collected the blood sample of ME/D immunity BALB/cByJ mouse the last time, and prepare serum according to standard method.Whether detect anti--ME/D antibody by ELISA exists.Shown in Fig. 8 B, detect the high titre IgG2a antibody that reacts with rME/A and rME/D, but do not have IgG1 antibody (Fig. 8 A).The existence of IgG2a antibody is the feature of Th1 type immunne response.F. in mouse, induce long-term memory to reply by recombination epitope and recombinant multi-epitope construction rME/D

The m tuberculosis infection that is determined at as described below is also replied with inductive long-term memory in recombination epitope rDNA5, rDNA9A, rDNA26, rDNA27, rDNA37, rDNA44 or rDNA45 or the recombinant multi-epitope construction ME/D immune mouse.In the mouse long-term memory is measured, with 10 of sublethal dose ⁴Colony-forming unit (CFU) mycobacterium tuberculosis injection BALB/cByJ mouse.After 4 weeks, treating 4 weeks of mouse again with microbiotic, to cure the m tuberculosis infection of mouse, then is the stationary phase in 8 weeks.Inject for the second time slip-knot nuclear mycobacterium (5 * 10 ⁵CFU) after 3 days, use above-mentioned splenocyte to measure and detect recombination to construct thing immunogenicity.Stimulate splenocyte with 2 μ M recombination epitopes or with 1,0.33,0.11,0.03 or 0.01 μ MrME/D.In Fig. 9 A-C, shown the IFN-γ generation level of in splenocyte is measured, measuring.Stimulate the splenocyte (Fig. 9 B) of control mice with irrelevant Protein G V14B.Other contrast in this experiment only comprises stimulates (Fig. 9 A) with substratum, 2 μ g/ml ConA, 10 μ g/ml PPD and 10 μ g/ml cow mycobacteriums.The memory T cell that reorganization ME/D stimulates the m tuberculosis infection mouse produces a large amount of IFN-γ (Fig. 9 B) in dosage dependence mode.In this measures, produce IFN-γ and show ME/D and the antigen of mycobacterium tuberculosis cross reaction of inducing permanent immunity to reply.As if be positioned at epi-position DNA5, DNA9A, DNA26, DNA27 and DNA44 upward (Fig. 9 B) with the antigenic determinant of antigen of mycobacterium tuberculosis cross reaction.

Embodiment 7 usefulness single epi-position of reorganization and recombinant multi-epitope construction stimulation human peripheral blood monocyte (PBMC)

Effect A. stimulation human peripheral blood monocyte (PBMC) in-vitro multiplication and secretion IFN-(IFN-γ)

Cultivate recombination epitope and recombinant multi-epitope construction and the human PBMC that the pET16 bacterial expression system is expressed in 37 ℃.After 48 hours, use enzyme-linked immunoassay (ELISA) to detect IFN-γ secretion as mentioned above.With the parallel culture of tritiate thymidine pulse, and use the synthetic PBMC of the evaluation propagation of DNA.These result of experiment are shown in Figure 10 A-B and Figure 11 A-B.The recombinant multi-epitope construction stimulates human PBMC's secretion of gamma-IFN and propagation (being respectively Figure 10 A and B).These reactions are dose-dependentlys, and than single recombination epitope inductive response intensity height (Figure 11 A and B).B. single epi-position of recombinating and recombinant multi-epitope construction rME/A and rME/D stimulate the external secrete cytokines of human PBMC

Following with the single epi-position of reorganization or reorganization rME/A or the external cytokine that stimulates the postevaluation human PBMC to produce again of rME/D.With 2 μ M recombinate single epi-position rDNA5, rDNA9A, rDNA26, rDNA27, rDNA29, rDNA37, rDNA44 or rDNA45, perhaps 0.5 μ M rME/A or rME/D irritation cell.Cellular control unit stimulates with 2 μ M GV14B or 10 μ g/ml PPD.Detect the cytokine reaction according to standard method with ELISA.As shown in table 7 below, the human PBMC who stimulates with the single epi-position of described reorganization or rME/A or rME/D produces Th1 cytokine IFN-γ and TNF-α.These recombination epitopes are secretion inducing IL-10 also.In the culture supernatant of irritation cell, do not detect Th2 cytokine IL-5.Low-level cytokine is secreted in the contrast antigenic stimulation, and is stimulating the back human PBMC to secrete the cytokine that institute tests to some extent with PPD.

Table 7

With human PBMC's excretory cytokine after single epi-position of reorganization and the rME stimulated in vitro

	Single epi-position of recombinating								??????????rME		Contrast
													Cytokine	??DNA ??5	??DNA ??9A	??DNA ??26	????DNA ????27	??DNA ??29	??DNA ??37	??DNA ??44	??DNA ??45	???rME/A	???rME/D	???GV14B	??PPD
?IFN-γ	??4.7	?2.8	??4.1	????0.06	??3.6	??4.3	??0.45	??0.78	????4.4	????3.2	????＜0.05	??3.96
													?TNF-α	??4.6	＜0.05	??1.2	????0.1	??3.5	??2.3	??0.5	??＜0.05	????3.9	????3.8	????0.2	??0.85
?IL-10	??0.75	＜0.05	??0.9	????0.15	??0.98	??0.83	??0.59	??＜0.05	????1.17	????1.12	????0.07	??0.34
													?IL-5	??0.08	＜0.05	??＜0.05	????0.06	??＜0.05	??＜0.05	??＜0.05	??＜0.05	????＜0.05	????＜0.05	????＜0.05	??0.06

Embodiment 8

With stimulating the macaque lymphopoiesis after the reorganization RME/D immunity

Three groups of macaques of immunity, 4 every group, with the immunogenicity of test recombinant multi-epitope construction rME/D.Detect lymphopoiesis with the PBMC proliferation assay.The macaque of I group is used PBS Freund's incomplete adjuvant (IFA) intradermal immunization of cumulative volume 0.1ml.The macaque of II group is with rME/D (the cumulative volume 0.1 ml) intradermal immunization of 33 μ g in IFA, and the macaque that III organizes is with rME/D (cumulative volume 0.1ml) intradermal immunization of 10 μ g in IFA.The 0th the week and the 6th week carried out twice immunity.Immunity 12 weeks of back are by every macaque blood-sample withdrawal.

The whole blood of every macaque was with dilution in 1: 5, and stimulate with control medium (adding the RPMI 1640 of 10% (v/v) autoserum, penicillin (60 μ g/ml), Streptomycin sulphate (100 μ g/ml) and glutamine (2mM)), described control medium comprises positive control phytohemagglutinin (PHA, 10 μ g/ml), PPD (10 μ g/ml), cow mycobacterium (10 μ g/ml), rME/D (1 μ g/ml) or contrast recombinant protein GV-14B (1 μ g/ml) in the total volume of culture of 1ml.Culture plate 72 hours, and then with the tritiate thymidine pulse in 1 μ Ci/ hole 18 hours, results also used scintillometer to measure the tritium picked-up.The lymphopoietic table 8 that the results are shown in.

The macaque blood sample that table 8PHA, PPD, cow mycobacterium, rME/D or rGV-14 stimulate drenches

Crust cell proliferation

Group	The macaque numbering	????PHA ??10μ/ml	????PPD ??10μ/ml	Cow mycobacterium 10 μ g/ml	??rME/D ??1μg/ml	??rGV-14 ??1μg/ml
							????1	????1	????18	????1.3	????0.73	????1.36	????0.77
	????2	????57	????2.3	????1.6	????2.4	????1.5
								????3	????32	????1.6	????1.5	????1.6	????1.0
	????4	????26	????2.2	????1.4	????1.6	????0.91
							????2	????1	????23	????1.6	????0.91	????15	????0.79
	????2	????70	????2.1	????2.0	????20	????2.2
								????3	????13	????1.0	????1.9	????12	????2.8
	????4	????40	????3.1	????2.5	????25	????3.3
							????3	????1	????35	????1.2	????1.9	????18	????2.4
	????2	????81	????1.0	????1.4	????32	????2.3
								????3	????29	????1.4	????2.2	????15	????1.3
	????4	????25	????0.89	????1.5	????1.3	????1.0

As shown in table 8, recombinant multi-epitope construction rME/D inductive PBMC cell proliferative response in the immune macaque of II group and III group is suitable with the proliferative response of positive control (PHA) inductive.With not recording propagation after PPD, cow mycobacterium or the rGV-14 stimulation.

Although in order to know that understanding the present invention has utilized drawings and Examples to describe in detail the present invention, but can under the situation that does not depart from the scope of the invention, the present invention be changed and modify, and the scope of the invention is only limited by the scope of appended claims.

Claims (25)

1. An isolated polypeptide comprising an amino acid sequence selected from SEQ ID NO: 61-77 sequences. 2. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a sequence having at least about 30% identical residues to any one of SEQ ID NOs: 61-63, 65, and 67-77, as determined by the computer algorithm FASTX; (b) a sequence having at least about 50% identical residues to any one of SEQ ID NOs: 61-63, 65, and 67-77, as determined by the computer algorithm FASTX; (c) a sequence having at least about 75% identical residues to any one of SEQ ID NOs: 61-77, as determined by the computer algorithm FASTX; (d) A sequence having at least about 90% residue identity with any one of SEQ ID NOs: 61-77, as determined by the computer algorithm FASTX. 3. An isolated polynucleotide encoding the polypeptide of any one of claims 1 and 2. 4. The isolated polynucleotide of claim 3, wherein said polynucleotide contains a sequence selected from the group consisting of SEQ ID NO: 8-21 sequences and complements thereof. 5. An isolated polynucleotide comprising a sequence selected from the group consisting of: (a) a sequence having at least about 30% identical residues to any one of SEQ ID NOs: 8-21, as determined by the computer algorithm BLASTN; (b) a sequence having at least about 50% identical residues to any one of SEQ ID NO: 8-21, as determined by the computer algorithm BLASTN; (c) a sequence having at least about 75% residue identity with any one of SEQ ID NO: 8-21, as determined by the computer algorithm BLASTN; (d) a sequence having at least about 90% residue identity with any one of SEQ ID NO: 8-21, as determined by the computer algorithm BLASTN; (e) The complement of a sequence of (a), (b), (c) or (d). 6. A DNA construct comprising at least one polynucleotide according to any one of claims 3-5. 7. The DNA construct of claim 6, wherein said construct comprises a sequence selected from SEQ ID NO: 56-58. 8. A host cell transformed with the DNA construct of any one of claims 6 and 7. 9. A fusion protein comprising at least one polypeptide according to any one of claims 1 and 2. 10. The fusion protein of claim 9, wherein said fusion protein comprises a sequence selected from SEQ ID NO: 79-81. 11. A composition comprising at least one polypeptide according to any one of claims 1 and 2 and a physiologically acceptable carrier. 12. A composition comprising at least one polypeptide according to any one of claims 3-5 and a physiologically acceptable carrier. 13. A composition comprising at least one fusion protein according to claim 9 and a physiologically acceptable carrier. 14. A composition comprising at least one DNA construct according to claim 6 and a physiologically acceptable carrier. 15. A composition comprising at least one polypeptide according to any one of claims 1 and 2 and an immunostimulator. 16. A composition comprising at least one polynucleotide according to any one of claims 3-5 and an immunostimulant. 17. A composition comprising at least one fusion protein of claim 9 and an immunostimulator. 18. A composition comprising at least one DNA construct of claim 6 and an immunostimulator. 19. A method of enhancing an immune response in a patient, the method comprising administering to said patient a composition according to any one of claims 11-18. 20. The method of claim 19, wherein the immune response is a Th1 response. 21. The method of claim 19, wherein said composition activates at least one cell selected from T cells and NK cells. 22. The method of claim 19, wherein said composition stimulates cytokine production. 23. The method of claim 19, wherein said composition induces long-term memory cells. 24. A method of treating a disease in a patient comprising administering to said patient the composition of any one of claims 11-18, wherein said disease is selected from the group consisting of immune disease, infectious disease and cancer. 25. The method of claim 29, wherein the disease is tuberculosis.

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